Detection of Opisthorchis viverrini Infection among the ASEAN Population in Thailand Using a Verbal Screening Test and Fecal Concentrator Kit

Background: Opisthorchis viverrini is a serious health problem in Southeast Asia. The infection is associated with cholangiocarcinoma. Therefore, this study was aimed to detect O. viverrini infections among the ASEAN population in Thailand. Methods: A cross-sectional study was conducted among 249 individuals from ASEAN populations in Thailand including Thai, Laotian, Cambodian, and Myanmar. Participants were screened using the O. viverrini verbal screening test (OvVST). Fecal samples were processed by the mini-parasep sf parasite fecal concentrator. Results: The infection rate of O. viverrini was 27.21%. The majority of infections was detected in females, in the age group 31–40 yr old, in the primary school education level, and in the occupation of labor. By country, O. viverrini infection was detected more often in the Lao PDR (30.77%). In screening for O. viverrini infection, OvVST had a high sensitivity (93.48%), specificity (86.70%), NPV (98.32%), and accuracy (87.95%). The PPV was 61.43% for OvVTS. The observed agreement was substantial for OvVST (k-value = 0.64). Conclusion: O. viverrini infections are still detected in ASEAN countries therefore large scale active surveillance is required. OvVST had a high sensitivity, specificity, and accuracy for screening the risk groups for O. viverrini.

Presently, the O. viverrini infection has been classified as Type 1 carcinogens by the International Agency for Research on Cancer, WHO (7). CCA is responsible for a major proportion of the burden of disease and death in Thailand, apart from causing hundreds of millions of people to surrender their rights to healthy and dignified lives (8). O. viverriniinduced CCA ranks first in mortality among cancers for men and second among cancers in women in the Mekong Basin sub-region (1,8,9).
In addition, O. viverrini-induced CCA is expected to increase sharply in the near future as a result of the demographic and economic factors occurring in Thailand, Lao PDR, Cambodia and Vietnam. The spread of liver fluke infection in the region due to increased migration among the ASEAN Economic Community (AEC) countries (Thailand, Laos, Cambodia, Vietnam and Myanmar) as a result of an open borders policy started in 2015 (8). For this reason, O. viverrini constitutes an important health problem in many parts of Southeast Asia and eradication of the fluke populations is urgently needed in these areas. Low cost and effective tool is needed for active surveillance among the risk group.
We aimed to screen the population at risk for O. viverrini and also detect the infection.

Materials and Methods
A cross-sectional study was conducted among the total of 249 participants including Thai, Cambodian, Laotian, and Burmese, who work or habitat in Nakhon Ratchasima province, in northeastern region of Thailand during August 2016 to February 2017, were enrolled.
All participants provided informed consent before participating in the study. This study was performed in accordance with good clinical practice and the guidelines of the Declaration of Helsinki. Ethical clearance was obtained from the Ethics Committee for Research Involving Human Subjects, Suranaree University of Technology (EC-59-39).
Populations at risk for O. viverrini infection were screened by the mini-verbal screening questionnaires; Ov verbal screening test: Ov-VST, through interviewed or self-checked using paper or mobile application. OvVST was created and literature reviewed from the basic knowledge that related to life cycle of O. viverrini, and then translated to Laotian and Cambodian language. For Burmese, translator was needed to translate for participants. Meanwhile, the participant could be self-checked by themselves through mobile application; SUT-OVCCA application in iOS and android platform (Fig. 1). OvVST is contained 1) the general information including gender, age, education, marital status, and nationality, and 2) the question with yes/no choices related to history of (i) consumption of raw spicy salad cyprinoid fish, (ii) consumption of raw minced cyprinoid fish, (iii) consumption of raw prickled cyprinoid fish, (iv) consumption of raw preserved small cyprinoid fish, (v) consumption of raw fermented cyprinoid fish, (vi) diagnosed as the opisthorchiasis, (vii) family member had diagnosed as the opisthorchiasis, O (viii) family member consumed the various dishes of raw cyprinoid fish, (ix) trend to consume the various cyprinoid fish, and (x) relative family had diagnosed a cholangiocarci-noma. The OvVST was tested and tryo ut before the study, and then analyzed for the reliability (Cronbach alpha coefficient was 0.75). Fecal specimens were collected, processed and examined following the manufacturer instructions for the mini-parasep sf fecal parasite concentrator, a new fecal parasite concentrator developed by the company DiaSys Europe Limited (formerly Intersep Ltd). The tubes and the sedimentation cones were labeled with the specimen identification numbers. A level fecal sample was introduced into each tube containing 3.3 ml of 10% formolsaline using the spoon on the end of the Mini Parasep SF filter. The MPFC was sealed by screwing in the filter/sedimentation cone unit. This was then vortexed for emulsification with the sedimentation cone pointing upwards. The MPFC was then inverted and centrifuged at 1,500 rpm for 2 minutes. The mixing chamber and the filter were then unscrewed and discarded for incineration while the supernatant in the sedimentation cone was decanted. The deposit was then examined microscopically using physiological saline and iodine for the eggs and larvae of intestinal parasites. Each of the preparations was examined systematically under the microscope for a minimum of 5 minutes. All preparations were initially screened with a low-power (10x) objective lens. Suspected parasitic objects were subsequently examined under a high-power (40x) objective.
Stool samples were examined by two laboratory technologists and then confirmed by an expert parasitologist. Finally, the data were analyzed and interpreted accordingly. Patients who were infected with other known parasites were treated with anti-parasitic drugs and also attended health education.
The helminthic eggs found in the fecal samples from the 249 participants, as well as their respective frequencies, are shown in Table 2, according to the diagnostic method used. From the total enrolled study participants 23.69% (59/249) were infected by one or more helminthic infection. By species the detected parasites were O. viverrini 18.47%, followed by Endolimax nana (1.61%), and hookworm (1. 16%).
The populations at risk for O. viverrini infection were classified into varying risk and no risk groups; the largest group had moderate risk (38.57%), followed by low risk (32.86%), and high risk groups (28.56%). The stool samples of the populations at risk for O. viverrini infection were examined and we found 43 positive cases and 27 negative cases. Positive cases were classified as moderate (20 cases), high risk (18 cases), and low risk (5 cases). Meanwhile, the no risk group of 179 participants was tested and only 3 were found to have O. viverrini on examination ( Table 3).
Fecal diagnosis results based on the fecal concentrator kit were used as the gold standard to estimate the sensitivity, specificity, NPV and PPV, and accuracy of the OvVST methods for screening the population at risk for O. viverrini infection ( Table 4). The parameters measured for OvVST were as follows; sensitivity (93.48%), specificity (86.70%), PPV (61.43%), NPV (98.32%), and accuracy (87.95%). The agreement of OvVST with the comparison between screening results and fecal detecting results was calculated by the MPFC method. The observed agreement was substantial for OvVST (k-value = 0.64, mean rank =0.51-0.74).

Discussion
ASEAN was founded in 1967 in order to promote economic and cultural development, to promote trade, industrial, agricultural, and scientific collaboration, and to promote peace and stability in the region (11). Ten member states make up ASEAN including Brunei Darussalam, Cambodia, Indonesia, the Lao PDR, Malaysia, Myanmar, the Philippines, Singapore, Thailand, and Viet Nam. The ASEAN Economic Community countries have had an open borders policy since 2015. These countries also harbor a mostly hidden burden of poverty and neglected tropical diseases (NTDs). The three major intestinal helminth infections are the most common NTDs; each type of helminthiasis is associated with approximately 100 million infections in the region. In addition, more than 10 million people suffer from either liver or intestinal fluke infections (12). Pullan et al. (13) in reporting on soiltransmitted helminth infections determined that 126.7 million people in Southeast Asia are infected with Ascaris roundworms, while 115.3 million are infected with Trichuris whipworms, and 77.0 million have hookworm infections (14,15). Thus, approximately onehalf of the people of Southeast Asia living in poverty have one or more soil-transmitted helminth infections.
In this study, 23.69% were infected by one or more helminthic infection. The majority of infections were O. viverrini, followed by hookworm, Endolimax nana, Strongyloides stercoralis, Taenia spp., Blastocystis hominis, and Entamoeba histolytica. This study shows a higher prevalence than a previous study. In Myanmar migrant workers in Thailand the overall prevalence of intestinal parasitic infections was 13.6%. The migrant workers were mainly infected with the fecal-oral transmitted parasites; E. histolytica / dispa (3.8%), A. lumbricoides (3.3%), and T. trichiura (2.3%) (16). The total infection rate was low among this group when compared to other studies. Sagnuankiat et al. (17) in reporting on the prevalence of intestinal parasitic infections among 372 immigrant children at 8 child-daycare centers during their parents' work time, by fecal examination found intestinal parasitic infections highly prevalent, at 71.0%. These infections comprised both helminths and protozoa: Trichuris trichiura (50.8%), Enterobius vermicularis The results indicate that migrant workers, particularly Laotian and Cambodian, should be large scale screened to prevent and control O. viverrini transmission. O. viverrini is a common parasite found in central and southern Laos and constitutes a major public health problem in the country. The Lao people continue to have the habit of extensively consuming raw or half-cooked fish, which are intermediate hosts. In Khammouane Province, the infection rate with O. viverrini was 54.8%. Factors associated with O. viverrini infections were gender, a habit of defecation in fields, and raw fish consumption (19). The overall liver/intestinal helminth egg positive rate was 71.9% among 6,178 residents in 9 provinces, Lao PDR. O. viverrini/minute intestinal fluke revealed the highest prevalence (55.6%); the endemic regions were Savannakhet, Khammouane, Vientiane (Nam Ngum), Champasak (Khong Island), and Saravane Province (20).  (26). The present study also indicates that O. viverrini is still a problem in Thailand, the Lao PDR, and Cambodia, but is not a serious problem in Myanmar.
The countries that comprise ASEAN have experienced impressive economic growth in recent years. However, such rapid growth has also left a substantial fraction of people economically marginalized. Overall, almost 200 million people in ASEAN countries, or roughly 30% of the population, live in extreme poverty, i.e., on less than US$2 per day, or below their national poverty lines (27)(28)(29). Countries also harbor a mostly hidden burden of neglected tropical diseases (NTDs). Of the almost 200 million people who live in extreme poverty in ASEAN countries, mostly in the low or lower middle-income countries of Indonesia, the Philippines, Myanmar, Viet Nam, and Cambodia, many of them are affected by at least one NTD. For this reason, OvVST was developed and created for iOS and android platforms, as well as paper questionnaires. Fecal diagnosis results based on the fecal concentrator kit were used as the gold standard. The observed agreement between screening results and fecal detecting results by the MPFC method was substantial for the OvVST (k-value = 0.64, mean rank =0.51-0.74). OvVST is able to identify the populations at risk for O. viverrini infection, as it successfully detected 43 positive cases but only had 3 false negative cases. Of 27 participants who had scores in the risk group but were negative for O. viverrini infection, they possibly had treatment before participating in the screening project or they consumed raw fish that did not contain the infective stage of the parasites. This indicates that using OvVST for screening for O. viverrini infection, is both very simple to answer and very fast to analyze by themselves. Participants took only about 2 minutes/person to answer the questions and they were then able to calculate their own risk using the test.
The impact of intestinal parasitic infections on public health is well known; they can spread from infected immigrant areas to uninfected areas via close contact and fecal-oral transmission from contaminated food and water. These results indicate that intestinal helminth infections are a serious public health problem.

Conclusion
OvVST is a simple and fast screening test with low cost. This tool may useful for O. viverrini screening for the large scale prevention and control of the spread of this liver fluke.